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Modulating protein activity with small molecules binding to cryptic pockets offers great opportunities to overcome hurdles in drug design. Cryptic sites are atypical binding sites in proteins that are closed in the absence of a stabilizing ligand and are thus inherently difficult to identify. Many studies have proposed methods to predict cryptic sites. However, a general approach to prospectively sample open conformations of these sites and to identify cryptic pockets in an unbiased manner suitable for structure-based drug design remains elusive. Here, we describe an all-atom, explicit cosolvent, molecular dynamics (MD) simulations-based workflow to sample the open states of cryptic sites and identify opened pockets, in a manner that does not require a priori knowledge about these sites. Furthermore, the workflow relies on a target-independent parameterization that only distinguishes between binding pockets for peptides or small-molecules. We validated our approach on a diverse test set of seven proteins with crystallographically determined cryptic sites. The known cryptic sites were found among the three highest-ranked predicted cryptic sites, and an open site conformation was sampled and selected for most of the systems. Crystallographic ligand poses were well reproduced by docking into these identified open conformations for five of the systems. When the fully open state could not be reproduced, we were still able to predict the location of the cryptic site, or identify other cryptic sites that could be retrospectively validated with knowledge of the protein target. These characteristics render our approach valuable for investigating novel protein targets without any prior information.

Diseases such as chronic pain with complex etiologies are unlikely to respond to single, target-specific therapeutics but rather require intervention at multiple points within a perturbed disease system. Such approaches are being enabled by the rise of computational methods to identify key points of intervention and by new screening techniques that focus on a relevant condition or phenotype, rather than a specific target. Here we apply an in silico network pharmacology approach to identify small-molecule compounds with the potential to selectively disrupt the structure of a chronic-pain specific disease network, which we validate using a novel phenotypic screen that recapitulates key aspects of neuronal and pain biology by measuring changes in neuronal excitability in native sensory neurons. The combination of network pharmacology with a phenotypic screen is a powerful approach; we show that hit rates increase from 26% to 42%. This represents a rational approach to the discovery of compounds with a poly-pharmacology based therapeutic value, which will be vital for the discovery of treatments for complex disease.

Summary Small conductance potassium (SK) ion channels define neuronal firing rates by conducting the after-hyperpolarization current. They are key targets in developing therapies where neuronal firing rates are dysfunctional, such as in epilepsy, Parkinson's, and amyotrophic lateral sclerosis (ALS). Here, we characterize a binding pocket situated at the intracellular interface of SK2 and calmodulin, which we show to be shared by multiple small-molecule chemotypes. Crystallization of this complex revealed that riluzole (approved for ALS) and an analog of the anti-ataxic agent (4-chloro-phenyl)-[2-(3,5-dimethyl-pyrazol-1-yl)-pyrimidin-4-yl]-amine (CyPPA) bind to and allosterically modulate via this site. Solution-state nuclear magnetic resonance demonstrates that riluzole, NS309, and CyPPA analogs bind at this bipartite pocket. We demonstrate, by patch-clamp electrophysiology, that both classes of ligand interact with overlapping but distinct residues within this pocket. These data define a clinically important site, laying the foundations for further studies of the mechanism of action of riluzole and related molecules.

Cholinergic hypofunction is associated with decreased attention and cognitive deficits in the central nervous system in addition to compromised motor function. Consequently, stimulation of cholinergic neurotransmission is a rational therapeutic approach for the potential treatment of a variety of neurological conditions. High affinity choline uptake (HACU) into acetylcholine (ACh)-synthesizing neurons is critically mediated by the sodium- and pH-dependent high-affinity choline transporter (CHT, encoded by the SLC5A7 gene). This transporter is comparatively well-characterized but otherwise unexplored as a potential drug target. We therefore sought to identify small molecules that would enable testing of the hypothesis that positive modulation of CHT mediated transport would enhance activity-dependent cholinergic signaling. We utilized existing and novel screening techniques for their ability to reveal both positive and negative modulation of CHT using literature tools. A screening campaign was initiated with a bespoke compound library comprising both the Pfizer Chemogenomic Library (CGL) of 2,753 molecules designed specifically to help enable the elucidation of new mechanisms in phenotypic screens and 887 compounds from a virtual screening campaign to select molecules with field-based similarities to reported negative and positive allosteric modulators. We identified a number of previously unknown active and structurally distinct molecules that could be used as tools to further explore CHT biology or as a starting point for further medicinal chemistry.

Regulation of transcription is fundamental to development and physiology, and occurs through binding of transcription factors to specific DNA sequences in the genome. CSL (CBF1/Suppressor of Hairless/LAG-1), a core component of the Notch signaling pathway, is one such transcription factor that acts in concert with co-activators or co-repressors to control the activity of associated target genes. One fundamental question is how CSL can recognize and select among different DNA sequences available in vivo and whether variations between selected sequences can influence its function. We have therefore investigated CSL-DNA recognition using computational approaches to analyze the energetics of CSL bound to different DNAs and tested the in silico predictions with in vitro and in vivo assays. Our results reveal novel aspects of CSL binding that may help explain the range of binding observed in vivo. In addition, using molecular dynamics simulations, we show that domain-domain correlations within CSL differ significantly depending on the DNA sequence bound, suggesting that different DNA sequences may directly influence CSL function. Taken together, our results, based on computational chemistry approaches, provide valuable insights into transcription factor-DNA binding, in this particular case increasing our understanding of CSL-DNA interactions and how these may impact on its transcriptional control.

Metabolism of xenobiotic and endogenous compounds is frequently complex, not completely elucidated, and therefore often ambiguous. The prediction of sites of metabolism (SoM) can be particularly helpful as a first step toward the identification of metabolites, a process especially relevant to drug discovery. This paper describes a reactivity approach for predicting SoM whereby reactivity is derived directly from the ground state ligand molecular orbital analysis, calculated using Density Functional Theory, using a novel implementation of the average local ionization energy. Thus each potential SoM is sampled in the context of the whole ligand, in contrast to other popular approaches where activation energies are calculated for a predefined database of molecular fragments and assigned to matching moieties in a query ligand. In addition, one of the first descriptions of molecular dynamics of cytochrome P450 (CYP) isoforms 3A4, 2D6, and 2C9 in their Compound I state is reported, and, from the representative protein structures obtained, an analysis and evaluation of various docking approaches using GOLD is performed. In particular, a covalent docking approach is described coupled with the modeling of important electrostatic interactions between CYP and ligand using spherical constraints. Combining the docking and reactivity results, obtained using standard functionality from common docking and quantum chemical applications, enables a SoM to be identified in the top 2 predictions for 75%, 80%, and 78% of the data sets for 3A4, 2D6, and 2C9, respectively, results that are accessible and competitive with other recently published prediction tools.

The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001, eLife digest During mitosis, a cell duplicates its DNA and then divides, ultimately generating two genetically identical daughter cells. In eukaryotes, the process of DNA duplication occurs at multiple sites throughout the genome: at each site, the antiparallel strands of the parental DNA separate and provide a template for DNA polymerase (Pol), the enzyme that synthesizes the two new DNA strands. Duplication of the DNA proceeds in both directions from each site through the polymerization of nucleotides to form new strands of DNA that are complementary to the template strands. However, since DNA polymerases can only polymerize nucleotides in one direction, the 5′ to 3′ direction, synthesis of the so-called leading strand proceeds continuously, whereas the other, lagging strand is synthesized in fragments. The task of duplicating the bulk of the DNA is shared between Pol δ, which is primarily responsible for synthesis of the lagging strand, and Pol ε, which fulfils the same role for the leading strand. However, Pols δ and ε cannot initiate DNA synthesis by themselves; short RNA-DNA chains called primers must also be paired to each template strand. Production of the primers requires the concerted action of two more enzymes: an RNA polymerase known as primase, and another DNA polymerase called Pol α. It is known that completion of the RNA-DNA primer requires Pol α to increase the length of the RNA segment by adding extra nucleotides, but the details of this process are poorly understood. Perera et al. combined crystallographic, biochemical and computational evidence to describe how Pol α first recognizes and then extends the RNA strand in the primer. They found that Pol α recognizes the particular shape of double helix—an A-form helix—that is formed by the DNA template and the RNA primer. The geometry of this helix prompts the Pol α enzyme to start adding nucleotides to the RNA in the primer. Perera et al. determined that once a full turn of double-helix DNA has been synthesized, Pol α is no longer in direct contact with the A-form helix, which causes the enzyme to disengage and terminate polymerization, leaving behind the now complete RNA-DNA primer. Perera et al. offer a new paradigm for understanding the initiation of DNA synthesis in eukaryotic replication. Their work suggests that Pol α has the ability to discriminate between different shapes of the primer-template helix, thus providing a mechanistic understanding of primer release. The spontaneous release of the primer offers a simple and elegant way to limit DNA synthesis by Pol α, a polymerase that is prone to error, and to make the RNA-DNA primer directly available for extension by Pol δ and Pol ε. DOI: http://dx.doi.org/10.7554/eLife.00482.002

Notch is a conserved signaling pathway that specifies cell fates in metazoans. Receptor-ligand interactions induce changes in gene expression, which is regulated by the transcription factor CBF1/Su(H)/Lag-1 (CSL). CSL interacts with coregulators to repress and activate transcription from Notch target genes. While the molecular details of the activator complex are relatively well understood, the structure-function of CSL-mediated repressor complexes is poorly defined. In Drosophila, the antagonist Hairless directly binds Su(H) (the fly CSL ortholog) to repress transcription from Notch targets. Here, we determine the X-ray structure of the Su(H)-Hairless complex bound to DNA. Hairless binding produces a large conformational change in Su(H) by interacting with residues in the hydrophobic core of Su(H), illustrating the structural plasticity of CSL molecules to interact with different binding partners. Based on the structure, we designed mutants in Hairless and Su(H) that affect binding, but do not affect formation of the activator complex. These mutants were validated in vitro by isothermal titration calorimetry and yeast two- and three-hybrid assays. Moreover, these mutants allowed us to solely characterize the repressor function of Su(H) in vivo.

Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.